Compositions and Methods for Soft Tissue Augmentation

ABSTRACT

The present invention provides compositions comprising isolated human collagen, isolated human elastin and a pharmaceutically acceptable carrier wherein the human elastin is substantially insoluble in water with a molecular weight greater than 100 kDa. The present invention further provides methods and kits for soft tissue augmentation.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/476,406, filed May 21, 2012, which is a continuation of U.S. patentapplication Ser. No. 12/220,420, filed on Jul. 24, 2008, which claimsthe benefit of U.S. Provisional Application No. 60/962,289, filed onJul. 27, 2007, the contents of each of which are incorporated herein byreference in their entirety.

FIELD OF THE INVENTION

This invention relates generally to compositions comprising isolatedhuman collagen and isolated human elastin, and generally related tomethods and kits for soft tissue augmentation using these compositions.

BACKGROUND OF THE INVENTION

Natural skin is composed of many elements, including dermal fibroblastsand keratinocytes, hair follicles, nerves and blood vessels.Extracellular matrix components of skin, which are responsible for thestrength, elasticity and turgor of native, healthy skin, includecollagens, elastin and glycosaminoglycans. Collagen molecules providethe bulk of the tensile properties of all connective tissues in thehuman body, including skin. Elastin is a very long-lived protein thatnonetheless breaks down in the skin of older individuals. Elastinbreakdown contributes to skin drooping and wrinkles Hydration isretained in skin by the presence of glycosaminoglycans, which act as“sponges” to retain water and provide skin with its natural turgor.Without these critical extracellular matrix components, skin becomesthin, wrinkled, and weak.

Various forms of injectable products have been developed for skin andother soft tissue augmentation. These products fall into synthetic and“natural” categories, wherein natural materials are derived from animalor human tissues. Synthetic materials that have been used as tissuebulking agents include silicone, oils and waxes, but these materialssuffer from healing complications and are very viscous and difficult toinject. Animal-derived materials that have been described include bovinecollagen in injectable forms. However, bovine collagen inducesoccasional immune reactions in recipients, due to the fact that bovinecollagens are not identical to human collagens and can serve as antigensfor immune reactivity. Other animal-derived extracellular matrixmaterials include hyaluronic acid that is derived from rooster combs.This material is quite viscous and also has the drawback of being ofnon-human origin. Additionally, various preparations of elastincurrently in use have the drawback of inducing calcification uponimplantation.

The compositions and methods of the present invention address theseproblems and fulfill a long felt need in the art.

SUMMARY OF THE INVENTION

The present invention provides compositions comprising isolated humancollagen, isolated human elastin and a pharmaceutically acceptablecarrier where the human elastin is substantially insoluble in water witha molecular weight greater than 100 kDa. The composition can compriseisolated human collagen derived from engineered vascular tissue orderived from micro-bead culture. The composition can comprise isolatedhuman elastin derived from engineered vascular tissue or native vasculartissue. The isolated human elastin can be cross-linked.

The compositions can include about 10-100 mg/ml of isolated humancollagen, more preferably about 30 mg/ml of isolated human collagen. Theisolated human collagen can have a molecular weight of about 100 toabout 500 kDa. The compositions can include about 2 to about 60 mg/ml ofisolated human elastin, preferably about 3 to 30 mg/ml of isolated humanelastin.

The compositions can further include isolated human glycosaminoglycans.The compositions can further include one or more active agents selectedfrom the group consisting of one or more anti-inflammatory agents,tissue formation agents, adipose tissue formation agents, anesthetics,antioxidants, heparin, epidermal growth factor, transforming growthfactor, transforming growth factor-β, platelet-derived growth factor,fibroblast growth factor, connective tissue activating peptides,β-thromboglobulin, insulin-like growth factors, tumor necrosis factors,interleukins, colony stimulating factors, erythropoietin, nerve growthfactors, interferons or combinations thereof. The compositions canfurther comprise one or more cells or tissues, preferably adipose tissueor dermal fibroblasts.

The present invention also provides dermal or subdermal fillersincluding isolated human collagen, isolated human elastin and apharmaceutically acceptable carrier where the human elastin issubstantially insoluble in water with a molecular weight greater than100 kDa.

The compositions can further include elastin isolated from humannon-frozen vascular tissue which is substantially insoluble in water.The compositions of the present invention do not induce calcification invivo.

The present invention also provides methods for soft tissue augmentationin a subject comprising, administering a composition comprising isolatedhuman collagen, isolated human elastin and a pharmaceutically acceptablecarrier wherein the human elastin is substantially insoluble in waterwith a molecular weight greater than 100 kDa. The method of the softtissue augmentation can improve conditions including, but not limitedto, lines, folds, wrinkles, minor facial depressions, cleft lips,correction of minor deformities due to aging or disease, deformities ofthe vocal cords or glottis, deformities of the lip, crow's feet and theorbital groove around the eye, breast deformities, chin deformities,augmentation; cheek and/or nose deformities, acne, surgical scars, scarsdue to radiation damage or trauma scars, and rhytids. The soft tissuecan be located in the pelvic floor, in the peri-urethral area, near theneck of the urinary bladder, or at the junction of the urinary bladderand the ureter. The method of soft tissue augmentation can increasetissue volume. The compositions may be injected into the skin or may beinjected underneath the skin. The compositions include insoluble elastinderived from human vascular tissue that does not induce inflammatory orimmune response and does not induce calcification.

The present invention also include kits and methods of using the kitsfor augmentation of a soft tissue. The present kits include isolatedhuman collagen, isolated human elastin and a pharmaceutically acceptablecarrier wherein the human elastin is substantially insoluble in waterwith a molecular weight greater than 100 kDa a syringe; a sterilewrapper surrounding said syringe and providing a sterile environment forsaid syringe and any other material/ and or reagents necessary. The kitscan also include agents selected from the group consisting of heparin,epidermal growth factor, transforming growth factor, transforming growthfactor-β, platelet-derived growth factor, fibroblast growth factor,connective tissue activating peptides, β-thromboglobulin, insulin-likegrowth factors, tumor necrosis factors, interleukins, colony stimulatingfactors, erythropoietin, nerve growth factors, interferons, osteogenicfactors and bone morphogenic proteins.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In the case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the results of a polyacrylamide gel showing the veryhigh levels of collagen purity in this preparation, as compared to thepurified bovine collagen control.

FIG. 2 illustrates the results of a polyacrylamide gel showingimmunoreactivity of isolated proteins with elastin antibody, showingthat elastin is isolated having molecular weights in the range ofapproximately 100 kDa or greater.

FIG. 3 illustrates the low degree of calcification of implanted elastinpreparations in a juvenile rat model showing that elastin isolatedaccording to the present invention results in calcification levels thatare indistinguishable from vehicle control.

FIG. 4 H&E and alizarin red staining of elastin compared to commercial,purified bovine elastin, implanted into juvenile rats showing thatcalcification of elastin that is isolated according to the method ofinvention is negligible, while calcification of bovine elastin isextensive. Panel A and Panel B show H&E stain of explanted tissues 21days after implantation of human elastin that was isolated according tothe present invention (“Humacyte”) (Panel A) and commercially obtainedbovine elastin (“Bovine”) (Panel B). Panel C and Panel D show alizarinred stain of explanted tissues 21 days after implantation of humanelastin that was isolated according to the present invention(“Humacyte”) (Panel C) and commercially obtained bovine elastin(“Bovine”) (Panel D).

FIG. 5 illustrate the results of H&E and alizarin red staining ofelastin compared to syngeneic rat aorta, implanted into juvenile rats,showing that implanted elastin calcification comparable to or less thanthat induced by syngeneic aorta. Panel A and Panel B show H&E stain ofexplanted tissues 21 days after implantation of human elastin that wasisolated according to the present invention (“Humacyte”) (Panel A) andsyngeneic rat aorta containing elastin (“Rat”) (Panel B). Panel C andPanel D show alizarin red stain of explanted tissues 21 days afterimplantation of human elastin that was isolated according to the presentinvention (“Humacyte”) (Panel C) and syngeneic rat aorta containingelastin (“Rat”) (Panel D).

FIG. 6 illustrates the results of H&E and alizarin red staining ofelastin compared to phosphate buffered saline carrier, implanted intojuvenile rats showing that implanted elastin calcification is comparableto or less than that induced by saline carrier. Panel A and Panel B showH&E stain of explanted tissues 21 days after implantation of humanelastin that was isolated according to the present invention(“Humacyte”) (Panel A) and carrier (“PBS”) (Panel B). Panel C and PanelD show alizarin red stain of explanted tissues 21 days afterimplantation of human elastin that was isolated according to the presentinvention (“Humacyte”) (Panel C) and carrier (“PBS”) (Panel D).

FIG. 7 illustrates the results of a polyacrylamide gel stained withcomassie blue for total protein showing that human collagen isolatedaccording to the present invention exhibit very high purity.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides compositions for the augmentation of skinand other soft tissues. Preferably, the compositions are formulated forinjection. The compositions are composed of extracellular matrixcomponents that are derived from vascular tissues, including, but notlimited to, collagens, elastin and glycosaminoglycans. The extracellularmatrix elements are combined in such a way as to improve theirsimilarity to human skin extracellular matrix components, and also toincrease their longevity in vivo and to minimize complications ofadministration. Deriving extracellular matrix from vascular tissuesproduces a “vascular-supporting” injectable formulation, whichencourages host blood vessels to infiltrate and support the injectedproduct. Such vascular-derived extracellular matrix compositions havethe advantage of incorporating more easily into the host, and ofstimulating the formation of nourishing blood vessels to the treatedskin or other soft tissue. The extracellular matrix components areentirely of human origin, and may be derived from engineered or fromnative tissues. Unlike other injectable formulations for skinaugmentation that contain only collagens or only animal-derivedhyaluronans, these formulations contain other human extracellular matrixcomponents that render them more similar to native, healthy human skin.

Soft Tissue Augmentation

Augmentation of soft tissue, such as skin, can be an important factor inrecovering from injury or for cosmetic purposes. For example, withnormal aging, skin may become loose or creases can form, such asnasal-labial folds. In the face, creases or lines may adversely affect aperson's self esteem or even a career. Thus, there has been a need forcompositions and methods that can diminish the appearance of creases orlines.

Further, there are situations in which loss of tissue can leave anindentation in the skin. For example surgical removal of a dermal cyst,lipoatrophy or solid tumor can result in loss of tissue volume. In othercases, injuries, such as gunshot wounds, knife wounds, or otherexcavating injures may leave an indentation in the skin. Regardless ofthe cause, it can be desirable to provide a dermal filler that canincrease the volume of tissue to provide a smoother or more evenappearance.

One example for needed support is dermal augmentation in the face wheredermal and subdermal volume is lost due to aging.

The term “soft tissue augmentation” includes, but is not limited to, thefollowing: dermal tissue augmentation; filling of lines, folds,wrinkles, minor facial depressions, cleft lips and the like, especiallyin the face and neck; correction of minor deformities due to aging ordisease, including in the hands and feet, fingers and toes; augmentationof the vocal cords or glottis to rehabilitate speech; hemostatic agent,dermal filling of sleep lines and expression lines; replacement ofdermal and subcutaneous tissue lost due to aging; lip augmentation;filling of crow's feet and the orbital groove around the eye; breastaugmentation; chin augmentation; augmentation of the cheek and/or nose;bulking agent for periurethral support, filling of indentations in thesoft tissue, dermal or subcutaneous, due to, e.g., overzealousliposuction or other trauma; filling of acne or traumatic scars andrhytids; filling of nasolabial lines, nasoglabellar lines and infraorallines. Moreover, the present invention can be directed to hard tissueaugmentation .The term “hard tissue” includes but is not limited tobone, cartilage and ligament.

The soft tissue can be located in the pelvic floor, in the peri-urethralarea, near the neck of the urinary bladder, or at the junction of theurinary bladder and the ureter.

The term “augmentation” means the repair, decrease, reduction oralleviation of at least one symptom or defect attributed due to loss orabsence of tissue, by providing, supplying, augmenting, or replacingsuch tissue with the compositions of the present invention. Thecompositions of the present invention can also be used to prevent atleast one symptom or defect.

Dermal fillers are used to fill scars, depressions and wrinkles Dermalfiller substances have various responses in the dermis from phagocytosisto foreign body reactions depending on the material (Lemperle et al.,Aesthetic Plast. Surg. 27(5):354-366; discussion 367 (2003)). One goalof dermal fillers is to temporarily augment the dermis to correct thesurface contour of the skin without producing an unacceptableinflammatory reaction, hypersensitivity reaction or foreign bodyreaction that causes pain, redness or excessive scar formation for aperiod of time.

The ideal material for human skin augmentation would include one or moreof the critical extracellular matrix elements that provide skin itsmechanical properties. These elements include collagen, elastin andglycosaminoglycans. In addition, to obviate immune responses, thesematerials should optimally be of human origin. Human materials will alsoinduce less inflammatory reaction than animal-derived materials, andhence will be likely to persist longer after injection into therecipient, thereby extending and improving the cosmetic effect of aformulation suitable for injection.

Many types of dermal filling procedures can benefit from the use of thecompositions of the present invention. The uses of the present inventionare designed (but not limited) to be used to provide increased volume ofa tissue that, through disease, injury or congenital property, is lessthan desired. Compositions can be made to suit a particular purpose, andhave desired retention times and physical and/or chemical properties.

Exemplary uses of compositions of this invention can be particularlydesirable to fill facial tissue (e.g., nasolabial folds), to increasethe volume of the dermis in the lips, nose, around the eyes, the earsand other readily visible tissue. Additionally, the compositions can bedesirably used to provide bulk to increase the volume of skin secondaryto excavating injuries or surgeries. For example, the site around adermal cyst can be filled to decrease the appearance of a dimple at thesite of surgery.

As such, the present invention provides methods of skin augmentation byadministering the extracellular matrix compositions of the invention toa subject in need thereof. Preferably, the methods improve skin wrinklesand/or increase skin volume. The subject or patient treated by themethods of the invention is a mammal, more preferably a human. Thefollowing properties or applications of these methods will essentiallybe described for humans although they may also be applied to non-humanmammals, e.g., apes, monkeys, dogs, mice, etc. The invention thereforecan also be used in a veterinarian context.

Extracellular Matrix Protein Compositions

The present invention provides compositions comprising isolated humancollagen, isolated insoluble human elastin and a pharmaceuticallyacceptable carrier. These compositions may include additional proteinsand active agents as described in further detail herein.

The compositions of the present invention which combine collagen withother elements of native skin, such as elastin, and in some embodiments,glycosaminoglycans, provide superior tissue augmentation, elasticity andturgor, as compared to compositions comprising a single extracellularmatrix component (e.g. collagen). These compositions comprising isolatedhuman collagen and elastin provide increased persistence in vivo ascompared to compositions comprising collagen alone, due to the improvedsimilarity of the collagen/elastin matrix to natural human skin.

Further, as the extracellular matrix components are derived from humanvascular tissues that are subjected to decellularization prior toisolation of extracellular matrix components, the compositions providelonger persistence and retention in vivo (due to less inflammatorybreakdown), and will be less prone to inflammation, calcification, andimmune reaction, than components derived from animal sources andisolated without a decellularization step.

With respect to calcification, this complication is known to exist forvarious purified forms of elastin, though the mechanism that causes thecalcification remains unclear (Lee, et al., American Journal ofPathology 2006; 168: 490-498; Daamenet al., Biomaterials 2005; 26:81-92; Hollinger et al., Calcified Tissue International 1988; 42:231-236; Urry et al., Calcified Tissue Research 1976; 21: 57-65).Competing hypotheses for elastin calcification advanced by those skilledin the art include the intrinsic nature of elastin pentapeptides toinduce calcification, the central role of metalloproteinases in inducingcalcification and the central role of microfibril impurities in elastincalcification. However, the precise cause of elastin calcification invivo remains unknown.

Collagen

The compositions of the present invention include an effective amount ofisolated human collagen and a pharmaceutically acceptable carrier.Preferably, the human collagen is derived from engineered tissue invitro and has a molecular weight of approximately 100 kDa toapproximately 500 kDa. Preferably, the compositions of the presentinvention comprise about 10 mg/mL-100 mg/mL of isolated human collagen,preferably about 15 mg/ml -70 mg/ml of isolated human collagen, morepreferably about 20 mg/m1-60mg/m1 of isolated human collagen and mostpreferably 30 mg/ml of isolated human collagen.

To produce isolated human collagen, human vascular cells are cultured invitro so as to maximize their production of collagenous matrix. This isaccomplished by a combination of carefully selected growth factors andculture medium components, combined with physical stimuli of cells (suchas stretching, shearing or stirring) to increase collagen matrixsynthesis (see, for example U.S. Pat. No. 6,537,567). This culturedtissue is then subjected to a decellularization process that removescellular components and leaves behind a mostly collagen-basedextracellular matrix (see, for example U.S. Pat. No. 6,962,814). Thecollagen in this matrix can then isolated by one of several methodsknown in the art.

The collagen derived as described above has several advantages overcollagen derived from native tissues or using previously-describedmethods to derive engineered collagen. Engineered tissues are derivedfrom cells that are banked and highly screened for infectious agents,which makes this material generally safer than materials derived fromcadavers. Also, the material is derived from vascular smooth musclecells, resulting in a “vascular-friendly” extracellular matrix materialthat supports the formation of nourishing blood vessels. Further, thismethod for collagen isolation incorporates a decellularization step,whereby cellular components and proteins are actively removed from thecollagen matrix. This provides a highly pure collagen matrix product (atleast 70-80% purity as determined by any assay known in the art, such asSDS PAGE analysis) and decreases the potential for immune reaction tonon-extracellular matrix components.

Elastin

The compositions of the present invention also include an effectiveamount of isolated human elastin and a pharmaceutically acceptablecarrier. Preferably, the compositions of the present invention comprisehuman elastin that is cross-linked and insoluble. Further, it ispreferable that the compositions of the present invention comprise humanelastin that has a molecular weight of approximately 100 kDa, and morepreferably greater then 100 kDa, as determined by any assay known in theart such as SDS PAGE analysis. Moreover, the compositions of the presentinvention comprise a particle size less than about 200 μm, preferablyless than about 100 μm, more preferably less than about 50 μm. Thecompositions comprise about 2-60 mg/ml of isolated human elastin,preferably 3-30 mg/ml of isolated human elastin. The isolatedcross-linked elastin is substantially insoluble in water, wherein thewater-soluble elastin content is in the range of 0.1-10 wt %, preferablyin the range of 0.1-8 wt %, more preferably in range of 0.1-6 wt %, morepreferably in the range of 0.1-4 wt %, more preferably in the range of0.1-2 wt % and most preferably in the range of 0.1-1wt %. Alternatively,the elastin is completely insoluble in water. In some embodiments, it ispreferable to have elastin with amino acid length which permits thepersistence of the protein in vivo.

To produce isolated human elastin, human vascular cells are cultured invitro so as to maximize their production of cross-linked elastin. Thecross-linked elastin will be insoluble and will permit the persistenceof the protein in vivo. While there are multiple reports of cellsproducing non-crosslinked tropoelastin monomers in culture, it is knownto be very difficult to stimulate the formation of cross-linked elastinfrom human vascular cells in vitro. However, the present inventionprovides culture conditions whereby creation of insoluble elastin isachieved, as documented by the presence of desmosine cross-links thatare specific to elastin. These tissues that contain elastin may then besubjected to a decellularization process (as described above forcollagen), after which the elastin is collected from the remainingmatrix using any one of several standard techniques known in the art.

The purity of elastin is typically assessed by the profile of aminoacids in the final product, and by the presence of desmosinecross-links, which are specific for cross-linked and insoluble elastin.The amino acid compositions of elastin from various species have beenreported (Starcher et al., Analytical Biochemistry 1976; 74: 441-447).In particular, it is known that alanine residue concentrations ofgreater than 200/1000 total residues, and valine residues of greaterthan 70/1000 total residues, are consistent with highly pure elastin(Daamen et al. , Biomaterials 2001; 22: 1997-2005). However, manymethods are reported for the isolation of purified elastin, and noconsensus has been reached regarding the optimal method for elastinisolation and implantation (Daamen, W. F., Hafmans, T., Veerkamp, J. H.,van Kuppevelt, T. H., “Isolation of intact elastin fibers devoid ofmicrofibrils”, Tissue Engineering 2005; 11: 1168-1176).

The elastin derived as described above has several advantages overprevious reports of elastin isolation. The elastin would be derived fromhuman, and not animal, origin. Since the cells used to produce theelastin are banked and derived from human vascular tissue, this willresult in lower immunogenicity in the human recipients. The elastin thusgenerated is of a “vascular” type, which should also promote theinfiltration of blood vessels into the treated area to support tissuereconstitution. The decellularization process, as with the collagenproduction, removes unwanted and potentially immunogenic cellularcomponents from the elastin matrix. This provides a highly pure elastinmatrix product (>70-80% purity and means that this elastin product has alower propensity for immune reaction, inflammation, and calcification,which are known complications of implantation of xenogeneic elastins.Preferably, the isolated human elastin of the present inventioncomprises desmosine cross-links. More preferably, the desmosinecross-links are present in insoluble elastin at a ratio of above 10,000picomoles per milligram of vascular tissue.

In addition to the methods described above, human elastin may also beisolated from native human blood vessels by means that ensure very highpurity, and thus minimize chances for immune reaction, inflammation, andcalcification. Indeed, calcification is one particular complicationassociated with elastin implantation, and hence having species matchingand highly pure formations, that will minimize inflammatory response,are important to cosmetic outcome and function.

An immune and inflammatory response can be measured by various assaysknown in the art such as, but not limited to, MHC-peptide tetramer,ELISPOT, intracellular cytokine assay. In general, a 10-50% increase inT-lymphocytes over the base line level (e.g., wild type normal state),preferably a 50% increase in T-lymphocytes, more preferably a 40%increase in T-lymphocytes, and most preferably a 30% increase inT-lymphocyte production indicates a significant immune response.

Calcification levels can be measured by various assays known in the artsuch as, but not limited to, atomic spectroscopy and H&E and alizarinred staining In general, 75-99% reduction in calcification, preferably80% reduction in calcification, more preferably a 90% reduction incalcification, most preferably 95% reduction in calcification, indicatessignificant reduction in calcification with the compositions of thepresent invention as compared to other control forms of elastin such aspurified bovine elastin (for example, from Elastin Products Co). Thatis, the compositions of the present invention do not induce significantcalcification, e.g., calcification greater than 25%, preferablycalcification between 5-20%, more preferably between 10 and 15% ascompared to the vehicle control (or the wild type normal state in asubject prior to administration). Alternatively, calcification levels ofelastin preparation are indistinguishable form vehicle control.

In this embodiment, human blood vessels are treated with adecellularization process that removes cellular components andglycosaminoglycans. Preferably, the human blood vessels are extractedfrom discarded human umbilical cords, but may be obtained from otherparts of the body, including the aorta or other major arteries or veins.

The blood vessels thus treated consist primarily of collagen andelastin. Collagen may be removed from such treated blood vessels by anyone of a number of methods known in the art, including autoclavetreatment, pepsin digestion, collagenase digestion, high salttreatments, alkali treatments, etc. In this way, collagen matrix isremoved and elastin is retained.

Glycosaminoglycans

The compositions of the present invention may also include an effectiveamount of one or more isolated human glycosaminoglycans and apharmaceutically acceptable carrier. Human glycosaminoglycans may beisolated from engineered tissues. Engineered tissues, grown inserum-containing medium, produce an extracellular matrix with a highercontent of glycosaminoglycans than corresponding native tissues. Thus,extracellular matrix synthesized during culture contains high quantitiesof glycosaminoglycans and is consequently more “watery” than nativetissues. Hence, engineered tissues are ideal for the production andisolation of glycosaminoglycans, which bind water and confer tissueturgor to connective tissues.

To produce isolated human glycosaminoglycans, human vascular cells arecultured in medium containing high serum (i.e. >10% by volume of serum),and after several weeks, tissues are removed from culture and treatedwith hyaluronidase or other glycosaminoglycan-cleaving enzymes.Supernatant from this digestion is collected, containing high molecularweight glycosaminoglycans that may be isolated using dialysis,centrifugation, or other techniques known in the art. Theseglycosaminoglycans may then be used to confer tissue turgor to a treatedarea.

In addition to the methods described above, human glycosaminoglycans andhuman hyaluronic acid may be derived from native vascular tissues.Native human blood vessels are treated with a protease such as pepsin orcollagenase, in order to break up confining, fibrillar extracellularmatrix. Preferably, the native blood vessels are extracted fromdiscarded human umbilical cords. Such protease pre-treatment exposesglycosaminoglycans and hyaluronans to aqueous solution and allowsswelling. Glycosaminoglycans and hyaluronans may then be collected fromvascular tissues by any of a variety of techniques known in the art,including treatment with hyaluronidase, detergent treatment, ortreatment with other enzymes that cleave glycosaminoglycan moieties.

The supernatant collected from this treatment can then be purified forhigh molecular weight glycosaminoglycans by any of a variety of methods,including dialysis, centrifugation, immune isolation and precipitation,etc. Preferably, the glycosaminoglycans have a MW greater than 100,000kDa.

Additional Active Agents

The compositions of the present invention may also include an effectiveamount of one or more active agents and a pharmaceutically acceptablecarrier. In some embodiments, it may be useful to include one or moreanti-inflammatory agents, tissue formation agents, anesthetics,antioxidants and the like, or combinations thereof.

Anti-inflammatory agents can include, but are not limited to, naproxen,sulindac, tolmetin, ketorolac, celecoxib, ibuprofen, diclofenac,acetylsalicylic acid, nabumetone, etodolac, indomethacin, piroxicam,cox-2 inhibitors, ketoprofen, antiplatelet medications, salsalate,valdecoxib, oxaprozin, diflunisal, flurbiprofen, corticosteroids, MMPinhibitors and leukotriene modifiers or combinations thereof

Agents that increase formation of new tissues at the site of applicationcan include, but are not limited to, fibroblast growth factor (FGF),transforming growth factor-beta (TGF-β), platelet-derived growth factor(PDGF) and/or fragments of angiotensin II (A-II) or combinations thereof

Anesthetics can include, but are not limited to, those used in caudal,epidural, inhalation, injectable, retrobulbar, and spinal applications,such as bupivacaine, lidocaine, benzocaine, cetacaine, ropivacaine, andtetracaine, or combinations thereof.

Antioxidants can include, but are not limited to, Vitamin C, Vitamin A,Vitamin E, β-carotene, superoxide dismutase, catalase, selenoenzymeglutathione peroxidase, ubiquinones/ubiquinols, thioredoxin reductase,propyl, octyl and dodecyl esters of gallic acid, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT) andnordihydroguaiaretic acid or combinations thereof.

Compositions used in the invention may additionally include one or morebiologically active agents to aid in the healing or regrowth of naturaltissue. For example, one may incorporate factors such as heparin,connective tissue activating peptides, β-thromboglobulin, insulin-likegrowth factors, tumor necrosis factors, interleukins, colony stimulatingfactors, erythropoietin, nerve growth factors, interferons, osteogenicfactors including bone morphogenic proteins, and the like.

Any drug or other agent which is compatible with the compositions andmethods of manufacture may be used with the present invention. Decisionsto use such drug or agent are typically made by the attending physicianbased on judgments about the injury or defect being repaired.

Methods of Isolating and Purifying Extracellular Matrix Proteins

There are numerous art recognized techniques that can be used to extractextracellular matrix components from native and engineered tissues.Specific enzymes that may be used to extract collagen and elastin matrixcomponents include, but are not limited to, collagenase; pepsin;trypsin; elastase; matrix metalloproteinases; dispase; serine proteases;other suitable proteases; high concentrations of salts such as NaCl orother salts; alkali treatment; acid treatment; Heat (for example,autoclaving, boiling, or baking); detergents (for example, SDS or CHAPS)and/or hypotonic treatment, (for example, water) or combinations ofthese treatments.

There are numerous art recognized techniques that can be used to isolateand purify the extracellular matrix components that are extracted fromthe engineered or native vascular tissues. Such methods may include, butare not limited to, centrifugation; salt precipitation of proteins suchas collagen; immunoprecipitation; antibody-mediated binding to beadsfollowed by cleavage to isolate matrix component; isolation based uponhydrophobicity/hydrophilicity (for example, extracting hydrophobicelastin by adhesion to hydrophobic substrate such as polystyrene);dialysis (to remove low molecular weight contaminants, enzymes, salt,acid, for example); drying; altering pH of solution to induceprecipitation of extracellular components; inactivation of enzymes thatwere used for isolation; and/or chromatographic methods (for example,polyacrylamide gel electrophoresis or high performance liquidchromatography that separate components based upon charge and molecularweight); or combinations of these treatments.

There are numerous art recognized techniques that can be used todecellularize engineered or native tissues prior to extracellular matrixisolation, in order to increase the purity of the extracted matrix,enhance its biocompatibility and persistence in vivo, and to ease theisolation of selected matrix components. In one example, aqueoushypotonic or low ionic strength solutions facilitate cell lysis inengineered and native tissues through osmotic effects. Such solutionsmay comprise deionized water or an aqueous hypotonic buffer (e.g., at apH of approximately 5.5 to 8, preferably approximately 7 to 7.5).Decellularization may be accomplished using a single decellularizationsolution, or the construct may be incubated sequentially in two or moresolutions. Another approach involves immersing the construct inalternating hypertonic and hypotonic solutions.

Preferred decellularization agents include, but are not limited to,salts, detergent/emulsification agents and enzymes such as proteases,and/or nucleases. Combinations of different classes of detergents, e.g.,a nonionic detergent such as Triton X-100(tert-octylphenylpolyoxyethylene) and an ionic detergent such as SDS(sodium dodecyl sulfate) may be employed. Preferably, one or moredecellularization solutions include Triton X-100, CHAPS(3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate), or SDS inphosphate buffered saline (PBS). Other suitable detergents includepolyoxyethylene (20) sorbitan mono-oleate and polyoxyethylene (80)sorbitan mono-oleate (Tween 20 and 80), sodium deoxycholate, andoctyl-glucoside. In certain preferred embodiments, various additivessuch as metal ion chelators, e.g., EDTA (ethylenediaminetetraaceticacid) and/or protease inhibitors are included in the decellularizationsolution. Suitable protease inhibitors for use in decellularizationsolutions include, but are not limited to, one or more of the following:phenylmethylsulfonyl-fluoride (PMSF), aprotinin, leupeptin, andN-ethylmaleimide (NEM).

Various enzymes that degrade cellular components may be included in thedecellularization solution. Such enzymes include nucleases (e.g., DNAsessuch as DNAse I, RNAses such as RNAse A), and phospholipases (e.g.,phospholipase A or C). Certain proteases such as dispase II, trypsin,and thermolysin may be of use in decellularization. Thedecellularization solution preferably includes a buffer. In general, apH between about 5.5 and 8.0, preferably between about 6.0 and 7.8, morepreferably between about 7.0 and 7.5 is employed. Preferred buffersinclude organic buffers such as Tris (hydroxymethyl) aminomethane(TRIS), (N-[2-hydroxyethyl]piperazine-N-[2-ethanesulfonic acid] (HEPES),etc. Buffers including sodium phosphate, citrate, bicarbonate, acetate,or glutamate may also be used.

Physical forces such as the formation of intracellular ice may beemployed as a primary means of accomplishing decellularization or toaugment the activity of decellularization solutions. One such approachreferred to as vapor phase freezing involves placing the construct ortissue in an appropriate solution, e.g., a standard cryopreservationsolution such as Dulbecco's Modified Eagle Medium (DMEM), 10%dimethylsulfoxide (DMSO), 10% fetal bovine serum (FBS) and cooling at aslow rate, e.g., 1-2° C. Multiple freeze-thaw cycles may be employed.Colloid-forming materials may be added to the solution to reduceextracellular ice formation while allowing formation of intracellularice. Appropriate materials include polyvinylpyrrolidone (10% w/v) anddialyzed hydroxyethyl starch (10% w/v).

Pharmaceutical Compositions and Modes of Administration

The compounds of the present invention are administered to a patient inthe form of a pharmaceutical composition. A compound that isadministered in a pharmaceutical composition is mixed with apharmaceutically acceptable carrier or excipient such that atherapeutically effective amount is present in the composition.

By “pharmaceutically acceptable” is meant a material that is notbiologically or otherwise undesirable, i.e., the material may beincorporated into a pharmaceutical composition administered to a patientwithout causing any undesirable biological effects or interacting in adeleterious manner with any of the other components of the compositionin which it is contained. When the term “pharmaceutically acceptable” isused to refer to a pharmaceutical carrier or excipient, it is impliedthat the carrier or excipient has met the required standards oftoxicological and manufacturing testing or that it is included on theInactive Ingredient Guide prepared by the U.S. Food and Drugadministration. “Pharmacologically active” (or simply “active”) as in a“pharmacologically active” derivative or analog, refers to a derivativeor analog having the same type of pharmacological activity as the parentcompound and approximately equivalent in degree.

The terms “effective amount” or “therapeutically effective amount”refers to an amount of the compound that is nontoxic and necessary toachieve a desired endpoint or therapeutic effect (e.g., act as a dermalor subdermal filler).

A variety of preparations can be used to formulate the compositions oractive agents of the present invention to render the most appropriatepharmaceutical compositions. Techniques for formulation andadministration may be found in “Remington: The Science and Practice ofPharmacy, Twentieth Edition,” Lippincott Williams & Wilkins,Philadelphia, PA. For human administration, preparations should meetsterility, pyrogenicity, general safety and purity standards as requiredby the FDA. Administration of the pharmaceutical composition can beperformed in a variety of ways, as described herein.

The active agent may be administered, if desired, in the form of a salt,ester, amide, prodrug, derivative, or the like, provided the salt,ester, amide, prodrug or derivative is suitable pharmacologically.Salts, esters, amides, prodrugs and other derivatives of the activeagents may be prepared using standard procedures known to those skilledin the art of synthetic organic chemistry and described, for example, byJ. March, Advanced Organic Chemistry: Reactions, Mechanisms andStructure, 4th Ed. (New York: Wiley-Interscience, 1992).

The amount of active agent (e.g., collagen, elastin, etc.) administeredwill depend on a number of factors and will vary from subject to subjectand depend on the particular drug administered, the particular disorderor condition being treated, the severity of the symptoms, the subject'sage, weight and general condition, and the judgment of the prescribingphysician. The minimum amount of drug is determined by the requirementthat sufficient quantities of drug must be present in a device orcomposition to maintain the desired rate of release over the givenperiod of application. The maximum amount for safety purposes isdetermined by the requirement that the quantity of drug present cannotexceed a rate of release that reaches toxic levels. Generally, themaximum concentration is determined by the amount of agent that can bereceived in the carrier without producing adverse histological effectssuch as irritation, an unacceptably high initial pulse of agent into thebody, or adverse effects on the characteristics of the delivery devicesuch as the loss of tackiness, viscosity, or deterioration of otherproperties.

The term “dosage form” denotes any form of a pharmaceutical compositionthat contains an amount of active agent sufficient to achieve atherapeutic effect with a single administration. When the formulation isan injection, the dosage form is usually one such injection. Thefrequency of administration that will provide the most effective resultsin an efficient manner without overdosing will vary with thecharacteristics of the particular active agent, including both itspharmacological characteristics and its physical characteristics.

The compositions of the present invention can also be formulated forcontrolled release or sustained release. The term “controlled release”refers to a drug-containing formulation or fraction thereof in whichrelease of the drug is not immediate, i.e., with a “controlled release”formulation, administration does not result in immediate release of thedrug into an absorption pool. The term is used interchangeably with“nonimmediate release” as defined in Remington: The Science and Practiceof Pharmacy, Nineteenth Ed. (Easton, Pa.: Mack Publishing Company,1995). In general, the term “controlled release” as used herein includessustained release and delayed release formulations.

The term “sustained release” (synonymous with “extended release”) isused in its conventional sense to refer to a drug formulation thatprovides for gradual release of a drug over an extended period of time,and that preferably, although not necessarily, results in substantiallyconstant blood levels of a drug over an extended time period.

The present formulations may also include conventional additives such asopacifiers, colorants, gelling agents, thickening agents, stabilizers,surfactants, and the like. Other agents may also be added, such asantimicrobial agents, to prevent spoilage upon storage, i.e., to inhibitgrowth of microbes such as yeasts and molds. Suitable antimicrobialagents are typically selected from the group consisting of the methyland propyl esters of p-hydroxybenzoic acid (i.e., methyl and propylparaben), sodium benzoate, sorbic acid, imidurea, and combinationsthereof.

Administration of a compound of the invention may be carried out usingany appropriate mode of administration. Thus, administration can be, forexample, oral, parenteral, topical, transdermal, transmucosal (includingrectal and vaginal), sublingual, by inhalation, or via an implantedreservoir in a dosage form.

Depending on the intended mode of administration, the pharmaceuticalformulation may be a solid, semi-solid or liquid, such as, for example,a tablet, a capsule, a caplet, a liquid, a suspension, an emulsion, asuppository, granules, pellets, beads, a powder, or the like, preferablyin unit dosage form suitable for single administration of a precisedosage. Suitable pharmaceutical compositions and dosage forms may beprepared using conventional methods known to those in the field ofpharmaceutical formulation and described in the pertinent texts andliterature, e.g., in Remington: The Science and Practice of Pharmacy(Easton, Pa.: Mack Publishing Co., 1995).

The dose regimen will depend on a number of factors that may readily bedetermined, such as severity of the condition and responsiveness of thecondition to be treated, but will normally be one or more doses per day,with a course of treatment lasting from several days to several months,or until a cure is effected or a diminution of disease state isachieved. One of ordinary skill may readily determine optimum dosages,dosing methodologies, and repetition rates. In general, it iscontemplated that the formulation will be applied one to four timesdaily. With a skin patch, the device is generally maintained in place onthe body surface throughout a drug delivery period, typically in therange of 8 to 72 hours, and replaced as necessary.

Preferably, the pharmaceutical compositions of the present invention canbe administered parenterally to a subject/patient in need of suchtreatment. The term “parenteral” as used herein is intended to includesubcutaneous (dermal or subdermal), intravenous, and intramuscularinjection or implantation (e.g., subcutaneously or intramuscularly or byintramuscular injection).

Preparations according to this invention for parenteral administrationinclude sterile aqueous and nonaqueous solutions, suspensions, andemulsions. Injectable aqueous solutions contain the active agent inwater-soluble form. Examples of nonaqueous solvents or vehicles includefatty oils, such as olive oil and corn oil, synthetic fatty acid esters,such as ethyl oleate or triglycerides, low molecular weight alcoholssuch as propylene glycol, synthetic hydrophilic polymers such aspolyethylene glycol, liposomes, and the like. Parenteral formulationsmay also contain adjuvants such as solubilizers, preservatives, wettingagents, emulsifiers, dispersants, and stabilizers, and aqueoussuspensions may contain substances that increase the viscosity of thesuspension, such as sodium carboxymethyl cellulose, sorbitol, anddextran. Injectable formulations are rendered sterile by incorporationof a sterilizing agent, filtration through a bacteria-retaining filter,irradiation, or heat. They can also be manufactured using a sterileinjectable medium. The active agent may also be in dried, e.g.,lyophilized, form that may be rehydrated with a suitable vehicleimmediately prior to administration via injection.

The quantity of active ingredient and volume of composition to beadministered depends on the host animal to be treated. Precise amountsof active compound required for administration depend on the judgment ofthe practitioner and are peculiar to each individual.

A minimal volume of a composition required to disperse the activecompounds is typically utilized. Suitable regimes for administration arealso variable, but would be typified by initially administering thecompound and monitoring the results and then giving further controlleddoses at further intervals.

A carrier for parenteral administration can be a solvent or dispersionmedium containing, for example, water, ethanol, polyol (for example,glycerol, propylene glycol, and liquid polyethylene glycol, and thelike), suitable mixtures thereof, and vegetable oils. The properfluidity can be maintained, for example, by the use of a coating, suchas lecithin, by the maintenance of the required particle size in thecase of dispersion and by the use of surfactants. The prevention of theaction of microorganisms can be brought about by various antibacterialand antifungal agents, for example, parabens, chlorobutanol, phenol,sorbic acid, thimerosal, and the like. In many cases, it will bepreferable to include isotonic agents, for example, sugars or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the use in the compositions of agents delayingabsorption, for example, aluminum monostearate and gelatin. It is alsoadvantageous to include one or more cells or tissues which maysupplement the use of the composition of the present invention. Forexample, it is preferred to include adipose tissue or cells, dermalfibroblasts or combination of thereof

Suitable preservatives for use in solution include benzalkoniumchloride, benzethonium chloride, chlorobutanol, thimerosal and the like.Suitable buffers include boric acid, sodium and potassium bicarbonate,sodium and potassium borates, sodium and potassium carbonate, sodiumacetate, sodium biphosphate and the like, in amounts sufficient tomaintain the pH at between about pH 6 and pH 8, and preferably, betweenabout pH 7 and pH 7.5. Suitable tonicity agents are dextran 40, dextran70, dextrose, glycerin, potassium chloride, propylene glycol, sodiumchloride, and the like, such that the sodium chloride equivalent of theophthalmic solution is in the range 0.9 plus or minus 0.2%. Suitableantioxidants and stabilizers include sodium bisulfite, sodiummetabisulfite, sodium thiosulfite, thiourea and the like. Suitablewetting and clarifying agents include polysorbate 80, polysorbate 20,poloxamer 282 and tyloxapol. Suitable viscosity-increasing agentsinclude dextran 40, dextran 70, gelatin, glycerin,hydroxyethylcellulose, hydroxmethylpropylcellulose, lanolin,methylcellulose, petrolatum, polyethylene glycol, polyvinyl alcohol,polyvinylpyrrolidone, carboxymethylcellulose and the like.

The compositions of the invention can be formulated for parenteraladministration by dissolving, suspending or emulsifying in an aqueous ornonaqueous solvent. Vegetable (e.g., sesame oil, peanut oil) or similaroils, synthetic aliphatic acid glycerides, esters of higher aliphaticacids and propylene glycol are examples of nonaqueous solvents. Aqueoussolutions such as Hank's solution, Ringer's solution or physiologicalsaline buffer can also be used. In all cases the form must be sterileand must be fluid to the extent that easy syringability exists. It mustbe stable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms, such asbacteria and fungi.

Solutions of active compounds as free base or pharmacologicallyacceptable salts can be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof

The preparation of more, or highly, concentrated solutions forsubcutaneous or intramuscular injection is also contemplated. In thisregard, the use of DMSO as solvent is preferred as this will result inextremely rapid penetration, delivering high concentrations of theactive compound(s) or agent(s) to a small area.

The present invention also provides kits for performing soft tissueaugmentation. Such kits can be prepared from readily available materialsand reagents and can come in a variety of embodiments. For example, suchkits can comprise, in an amount sufficient for at least one treatment,any one or more of the following materials: human elastin and collagenisolated by methods of the present invention, sterilized buffers (e.g.,phosphate buffered salt) or water, other reagents necessary or helpfulto perform the method, and instructions. Typically, instructions includea tangible expression describing reagent concentration or at least onemethod parameter, such as the amount of reagent to be used, maintenancetime periods for reagents, and the like, to allow the user to carry outthe methods described above. In a preferred embodiment of the invention,a kit comprises a means for delivery. Such means can include, by way ofillustration and not limitation, a small syringe (22 to 27-gauge), alarge syringe (13 to 19-gauge) and equipment used in endoscopic orpercutaneous discectomy procedures. The reagents can be provided insolution, as suspensions, or as a substantially dry powder, e.g., inlyophilized form, either independently or in a mixture of components toimprove ease of use. Where a degradable reagent is provided, conditionsare chosen so as to stabilize the reagent, e.g., storage at lowertemperature, addition of stabilizing agents (e.g., glycerol or areducing agent). Unstable reagents can be provided together with orseparately from the more stable components of the kit.

While the invention has been described in conjunction with the detaileddescription thereof, the foregoing description is intended to illustrateand not limit the scope of the invention, which is defined by the scopeof the appended claims. Other aspects, advantages, and modifications arewithin the scope of the following claims.

The present invention is further illustrated by the following examplesthat should not be construed as limiting in any way.

EXAMPLES Example 1 Isolation of Collagen from Engineered Vascular Tissue

Vascular tissues are engineered from human vascular smooth muscle cellsaccording to methods as previously described (Niklason et al., Science284(5413):489-93, 1999). Briefly, vascular smooth muscle cells fromscreened and banked human vascular cell sources are seeded onto atubular synthetic fibrous scaffolding comprising polyglycolic acidfibers. The tubular seeded scaffold is threaded over distensiblesilicone tubing within a sterile bioreactor. The bioreactor is filledwith culture medium that supports the synthesis of collagen by vascularcells. Specifically, this medium comprises Dulbecco's Modified EaglesMedium (DMEM) supplemented with 20% fetal bovine serum or other serum,ascorbic acid (50 mg/L), growth factors such as platelet derived growthfactor (10 ng/mL), basic fibroblast growth factor (10 ng/mL), epidermalgrowth factor (3 ng/mL), proline 50 mg/L, glycine 50 mg/L, alanine 20mg/L, copper sulfate 3 ng/mL. Other medium components that supportgrowth of cells and/or extracellular matrix production may also beincluded in the culture medium. Cyclic pulsatile radial strain may beadministered to the tubular constructs over the silicone tubing bypumping fluid through the tubing, with distensions of 1-5% being mostpreferable. Alternatively, cyclic strain may be omitted during culture,in order to simplify the culture system. Culture is maintained for 2-10weeks, during which time collagenous matrix is synthesized by thevascular smooth muscle cells.

At the conclusion of culture, the engineered vascular tissue isdecellularized using techniques similar to those reported in the art(Dahl, Cell Transplant 12(6):659-66, 2003). Specifically,detergent-based decellularization can incorporate two differenttreatment solutions. Solution 1 includes 8 mM CHAPS, 1.0 M NaC1, and 25mM EDTA in PBS. Engineered vascular tissues are exposed to this solutionfor one hour. Following rinses in PBS, engineered vascular tissues arethen exposed to Solution 2 for one hour. Solution 2 includes 1.8 mMsodium dodecyl sulfate, 1.0 M NaC1, and 25 mM EDTA in PBS. Engineeredvessels are then rinsed in PBS and are rendered acellular by thisprocess. All treatments are performed at room temperature or at about37° C.

The following steps are used to isolate and purify the collagen from thedecellularized, engineered human vascular tissues:

1. Begin with a cellular engineered material. Cut, slice, blend, chopinto small pieces.

2. Digest tissue material in pepsin (0.5 to 2.0 mg/ml pepsinconcentration dissolved in a low pH solution) at 4-20° C. Agitationduring digest will aid the process.

3. Once digestion has completed, centrifuge briefly to remove anyundigested material.

4. Remove supernatant and raise the pH of the solution to about pH 8.5by slowly adding NaOH to inactivate pepsin.

5. Using HC1, bring the pH of the solution back to about pH 3.5.

6. Clarify collagen solution using diatomaceous earth.

7. Precipitate clarified collagen by adding NaCl to the solution.Precipitate at 4° C. for >24hrs.

8. Collect precipitated collagen by chilled centrifugation at highspeeds for ˜30 minutes.

9. Aspirate supernatant carefully and resuspend precipitated collagensin ice-cold HCl. Allow for collagen molecules to completely solubilized.

10. Dialyze solution to further purify collagen.

11. Concentrate collagen to desired level.

12. Store this purified collagen in solution.

13. Add sterile-filtered sodium diphosphate solution to concentrated,purified, collagen, until final concentration of about 20-50 mM andabout pH 7.4 is reached. Incubate at 22-37° C. for >24 hours.

14. An opaque white fibrous precipitate will form, containing largemacromolecular collagen fibrils.

15. Centrifuge to obtain the resulting high concentration of fibrillarcollagen for injection, discarding supernatant.

Using these steps, collagen is extracted from engineered, decellularizedtissues. Extracted collagen is then run on a polyacrylamide gel toassess preservation of chain morphology and purity. FIG. 1 shows thevery high levels of collagen purity in this preparation, as compared tothe purified bovine collagen control. Hence, the methods in this exampleproduce highly pure, collagen from engineered vascular tissues.

Example 2 Isolation of Elastin from Engineered Vascular Tissue

Synthesis of cross-linked, insoluble elastin in cultured cells istypically quite difficult. While many reports exist of synthesis ofnon-crosslinked tropoelastin monomers, creation of documented,cross-linked elastin is extremely rare. Most reports of production ofinsoluble elastin utilize cells that have been genetically engineered,for example to express high levels of tropolelastin protein, or toexpress variants of versican that stimulate elastin deposition.Alternatively, rodent cells derived from neonatal animals have beenreported to synthesize elastin. However, non-human elastin is associatedwith risks of immune rejection if injected into a human recipient.Further, utilizing genetically modified cells to generate human elastincarries intrinsic risks of passing on transgene material to any eventualrecipient of the extracellular matrix material.

Hence, it is advantageous to devise methods to generate crosslinked,insoluble elastin from cultured human cells, without the use of geneticengineering. In particular, the use of human vascular cells that arebanked and have been screened for infectious agents also helps to reduceany infectious risk of resultant elastin that is produced.

The methods of the present invention culture human vascular tissues toproduce measurable amounts of insoluble, crosslinked elastin, asindicated by desmosine anlaysis. Such tissues may then be decellularizedand the elastin within these tissues extracted and purified. In oneexample, human vascular smooth muscle cells are seeded onto polyglycolicacid scaffolds in bioreactors, analogously to the procedure described inExample 1. Cyclic strain may be applied via the luminal silicone tubing,or may be omitted, in order to simplify the culture conditions. Totalculture time may vary from 2-10 weeks. In this example, total culturetime is 3 weeks. Culture medium that is designed to stimulate elastinsynthesis for the first two weeks of culture contains the followingcomponents:

1. 400 mL DMEM-low glucose

2. 100 mL Human Serum

3. 5 mL (50,000 U) of Penicillin G

4. 2.5 mg Insulin,

5. 0.5 μg CuSO₄

6. 5ml aliquot of Glycine/Alanine/Valine/Proline (30 mg/18 mg/17.5mg/11.5 mg)

7. Dexamethasone 10⁻⁸-10⁻¹⁰ M

8. 2.5 μg TGF-beta

In order to further stimulate elastin synthesis by cultured humanvascular smooth muscle cells within the engineered tissue, the followingmedium is used during the final week of culture:

1. 475 mL DMEM-low glucose

2. 25 mL Human Serum

3. 5 mL (50,000 U) of Penicillin G

4. 2.5 mg Insulin, (5 μg/ml)

5. 1.5 μg CuSO₄

6. 5m1 aliquot of Glycine/Alanine/Valine/Proline (30 mg/18 mg/17.5mg/11.5 mg)

7. Dexamethasone 10⁻⁸-10⁻¹⁰ M

8. 2.5 μg TGF-beta (5 ng/mL)

Using these culture conditions, engineered vascular tissues containingelastin, cells and other matrix components are generated. At theconclusion of culture, engineered vascular tissues are subjected to thedecellularization process as described in Example 1. Subsequent to thisdecellularization process, the intact and decellularized tissues aresubjected to analysis for desmosine, which is a covalent cross-linkcomponent in mature elastin. Presence of desmosine indicates theproduction of mature elastin by engineered vascular tissues. Table 1contains desmosine results for a variety of engineered vascular tissues,both intact and decellularized.

TABLE 1 pmol Desmosine/ pmol Desmosine/ Samples mg Protein mg TissueFresh-1 32 25 Fresh-2 29 27 Fresh-3 37 20 Fresh-4 49 35 Fresh-5 57 26Fresh-6 67 29 Fresh-7 41 12 Decellutarized-1 53 29 Decellutarized-2 3134 Decellutarized-3 39 19 Decellutarized-4 55 46 Decellutarized-5 37 25Decellutarized-6 28 35 Decellutarized-7 64 51

Table 1 shows that desmosine elastin cross-links are present in intactengineered tissues, and are retained after the decellularizationprocess. Hence, it is feasible to engineer cross-linked elastin that isstable following removal of cellular constituents. Isolation of elastinfrom engineered and decellularized vascular tissues may then beaccomplished by any one of several methods known in the art, including,but not limited to, autoclaving, alkali or acid treatment, pepsin orcollagenase digestion, or combinations of these treatments.

Example 3 Isolation of Elastin from Native Human Umbilical Vessels

Elastin is isolated from native human blood vessels, such as thoseisolated from discarded umbilical cords. Vessels are decellularized andthen elastin is isolated from the resultant extracellular matrix. Thesolutions for decellularization process are those described inExample 1. Steps used in the decellularization and elastin isolationprocess are as follows:

1. Thaw frozen umbilical artery or vein overnight at 4° C.

2. Record vessel length and weight.

3. Decellularize vessel in Solution 1 for 12 hr.

4. Rinse 2 times with phosphate buffered saline, 5 min each.

5. Decellularize in Solution 2 for 12 hr.

6. Rinse 3 times with phosphate buffered saline, 5 min each.

Digest decellularized umbilical artery or vein with pepsin orcollagenase at 37° C. or room temperature for 1-5 hr with gentle shakingThis removes non-elastin extracellular matrix components.

7. Alternatively, decellularized vessels may be autoclaved for 3-4cycles at 121° C. for 30 min or 115° C. for 20 min, to removenon-elastin extracellular matrix components.

8. Rinse digested tissue or autoclaved tissue with distilled water.

9. Snap freeze tissue in dry ice.

10. Lyophilize tissue overnight.

11. Subject resultant elastin to amino acid analysis to evaluate purity.

12. Further digest the purified elastin with pepsin to produceinjectable particle size.

13. Alternatively, mechanically break down elastin using mortar/pestleor a mill or homogenizer, to produce particles of a size appropriate forinjectable products, preferably less than about 200 μm, more preferablyless than about 100 μm, most preferably less than about 50 μm.

Human umbilical vessels are treated according to the steps in thisexample, and are analyzed for amino acid content to determine elastinpurity (step 12 above). Table 2 indicates the exact preparation stepsfrom the above list that are used for each sample.

TABLE 2 Samples 1 2 3 4 5 6 7 8 NaCI extraction y y Autoclave 115C 115C115C 115C 121C 121 Delipid Y y y y y y decell y y y Pepsin 37C RT

Table 3 shows results of the amino acid analysis of the resultantpurified elastins.

TABLE 3 Values are Residues/1000 Sample # 1 2 3 4 5 6 7 8 Expected *CVS16 0 24 2 23 3 0 0 3 asx 93 73 92 88 96 38 94 79 2 thr 38 23 45 42 43 2138 28 14 ser 31 24 34 31 34 13 37 30 9 glx 111 98 120 116 125 51 100 883 pro 87 104 76 84 68 113 94 144 129 gly 202 352 145 157 134 286 273 326312 ala 96 120 100 107 99 197 97 110 239 val 73 37 76 81 78 123 55 41137 *met 6 0 5 14 0 0 0 0 0 ile 44 24 48 53 50 31 52 28 24 leu 76 40 8584 88 66 69 45 65 *tyr 0 0 3 2 0 0 0 0 23 phe 28 15 33 34 35 26 29 20 24his 1 0 8 6 23 0 0 0 0 lys 50 36 60 57 59 25 33 29 9 arg 47 54 46 43 448 30 32 9

In Table 3, the “Expected” column indicates the number of amino acidresidues per 1000 amino acid residues that would be expected in the caseof completely pure human elastin. It is clear from Table 3 that Sample 6contains the most highly pure isolated elastin. This sample was preparedusing decellularization and autoclave extraction (see Table 2). Hence,in contrast to previously reported techniques that claim to isolate pureelastin from other types of tissues, it had been found that humanvascular tissues require an additional decellularization step in orderto isolate elastin of sufficient purity. This finding is in contrast tomultiple reports in the literature that claim that autoclave treatmentalone, when applied to other tissues such as bovine ligamentum nuchae,produces a highly pure elastin preparation (Lee et al., American Journalof Pathology 2006; 168: 490-498). From Table 3, it is clear thatstandard methods such as autoclaving, without an additionaldecellularization step, produce highly impure elastin products whenapplied to native human umbilical cord vascular material.

Example 4 Isolation of Elastin from Native Human Aorta

Elastin is also purified from human aorta. The process involves asalt-based decellularization step, followed by boiling in 0.1 N NaOH andthen extraction in hydrophobic solvents. Elastin isolation according tothis example has unexpected properties when implanted in vivo, as shownin Example 5 (below). Steps for purifying elastin from aorta accordingto the present invention are as follows:

Obtain wet weight of aorta. Aorta is preferably fresh or non-frozen.

1. Shred aorta using a blender or some other device in distilled water.

2. Extract shredded tissues at 1-hour intervals in 0.9% NaCl solution at4° C. with shaking

3. Repeat NaCl extraction until protein assay shows no soluble proteinextraction.

4. Suspend samples in boiling 0.1 N NaOH solution, boil for 40-45minutes.

5. Discard NaOH solution, then rinse with distilled water.

6. Extract elastin 3 times, 30 minutes each, with 100% ethanol at roomtemperature.

7. Extract elastin in 50% ethanol/50% diethyl ether for 1 hour at roomtemperature.

8. Extract elastin in 100% diethyl ether for 1 hour at room temperature.

9. Decant ether, dry overnight. Obtain final weight.

10. Grind or pulverize to create injectable and insoluble elastinparticles.

The amino acid analysis is performed, along with the RIA analysis fordesmosine cross-links, of elastin that is purified from human aortausing the above method. For these experiments, a total of 6 differentaortas are treated using this protocol, and amino acid analysis isperformed on 4 of the 6 samples. Desmosine quantification is performedon all samples as summarized in Table 4.

TABLE 4 AA analysis and Desmosine for elastin from human aorta: (per1.000 total residues) Amino Acid Sample 54 Sample 55 Sample 56 Sample 57Expected *cys 0 0 3 asx 7 7 5 6 2 thr 9 8 5 5 14 ser 6 6 3 3 9 glx 21 2120 19 3 pro 116 116 111 111 129 gly 332 331 353 353 312 ala 263 263 261259 239 val 114 116 127 127 137 *met 0 0 0 0 0 ile 23 25 21 21 24 leu 6061 55 58 65 *tyr 13 14 5 3 23 phe 24 25 19 21 24 his 0 0 0 0 0 lys 6 711 9 9 arg 5 4 5 5 9 Des 12332 13291 19793 11904 (pM/mg) *Cys, Tyr andMet are partially destroyed during acid hydrolysis Desmosine units arein (pico Moles/mg Protein)

As shown in Table 4, the values of alanine are well above 200 residuesper 1,000 total residues, and values of valine are well above 70residues per 1,000 residues. These are consistent with high purityelastin protein. In addition, values of Desmosine cross-links are veryhigh, and are comparable to or higher than those reported for elastinpreparations from a variety of species (see Table 5):

TABLE 5 Desmosine from aortas of different species (pM/mg protein)picomole/mg protein Cow 12573 Pig 13934 Monkey 10948 Rat 6266 Dog 12942

An alternative method in accordance with the present invention is toisolate elastin from human aorta using a pepsin digestion. FIG. 2 showsan immunoblot of proteins isolated from human aorta using pepsindigestion, and reacted with anti-elastin antibody. For digestion timesranging from 2-5 hours, extensive protein is liberated that reacts withelastin antibody, having molecular weights (MW) in the approximate rangeof 100-500 kDa (i.e., greater than 100 kDa).

Example 5 Implantation of Purified Elastin In Vivo

One drawback of other elastin preparations is a tendency to calcify invivo. Implantation into juvenile (i.e. 21 day-old) rats is an extremelysensitive assay for calcification. In order to determine the propensityof human elastin that is isolated according to the present invention tocalcify, the elastin was implanted subdermally into rats. The humanelastin was isolated from aorta according to the present invention usingsalt-based decellularization followed by NaOH extraction as described inExample 4. Calcification was compared to that induced by purified bovineelastin (purchased commercially), syngeneic rat aorta (containing ratelastin), and injection of phosphate buffered saline control. Implantsremained in situ for 21 days and then were explanted. Calcification wasassessed histologically, using the alizarin red stain, which produces areddish-brown color in calcified tissues. In addition, calciumaccumulation at implant sites was determined quantitatively by atomicabsorption spectroscopy.

FIG. 3 shows the atomic absorption spectroscopy of calcium content ofexplanted tissues from juvenile rats that contained various elastinimplants or control implants. Error bars are standard deviation of themean. Various samples were analyzed: juvenile rat subcutaneous tissue,negative control (SubCut Tissue); phosphate buffered saline carrier(PBS); syngeneic rat aorta, containing syngeneic rat elastin (RatElastin); human elastin isolated according to the present invention,from fresh aorta (E56); human elastin isolated according to the presentinvention, from fresh aorta, sterilized by gamma radiation (E56 gamma);human elastin isolated according to the present invention, from frozenaorta (E59); human elastin isolated according to the present invention,from frozen aorta, sterilized by gamma radiation (E59 gamma); purifiedbovine elastin obtained from Elastin Products Co. (B-elastin); purifiedbovine elastin from Elastin Products Co, sterilized by gamma radiation(B-elastin gamma); injectable form of bovine elastin obtained fromElastin Products Co. (B-elastin Injection); injectable form of bovineelastin obtained from Elasin Products Co, sterilized by gamma radiation(Injection B-elastin Gamma); and phosphate buffered saline carrier(Injection PBS).

The results of atomic absorption spectroscopy show that calcium levelsin explants having elastin that is isolated from non-frozen human aortaaccording to the present invention are not different from calcium levelsin tissues that are injected with PBS carrier. However, the results showthat when elastin was isolated according to the present invention fromfrozen aorta, tissue calcification was significantly increased. Overall,there does not appear to be any impact of sterilization by gammairradiation on the degree of tissue calcification for any of the formsof elastin that is tested (see FIG. 3 for the atomic absorptionspectroscopy results and Table 6 for summary of the correspondingquantitative values of calcium in tissue explants)

FIG. 4 shows the staining of explanted tissue specimens from juvenilerats implanted with elastin that was isolated according to the presentinvention, and with bovine elastin. A,B: Hematoxylin & eosin (H&E) stainof explanted tissues 21 days after implantation of human elastin thatwas isolated according to the present invention (“Humacyte”) andcommercially obtained bovine elastin (“Bovine”). C,D: Alizarin red stainof explanted tissues 21 days after implantation of human elastin thatwas isolated according to the present invention (“Humacyte”) andcommercially obtained bovine elastin (“Bovine”). Arrows in panel Dindicate areas of visible calcification.

FIG. 5 shows the staining of explanted tissue specimens from juvenilerats implanted with elastin that was isolated according to the presentinvention, and with syngeneic rat elastin from rat aorta. A,B:Hematoxylin & eosin (H&E) stain of explanted tissues 21 days afterimplantation of human elastin that was isolated according to the presentinvention (“Humacyte”) and syngeneic rat aorta containing elastin(“Rat”). C,D: Alizarin red stain of explanted tissues 21 days afterimplantation of human elastin that was isolated according to the presentinvention (“Humacyte”) and syngeneic rat aorta containing elastin(“Rat”). Arrows in panel D indicate areas of likely calcification.

FIG. 6 shows the staining of explanted tissue specimens from juvenilerats implanted with elastin that was isolated according to the presentinvention, and with phosphate buffered saline carrier. A,B: Hematoxylin& eosin (H&E) stain of explanted tissues 21 days after implantation ofhuman elastin that was isolated according to the present invention(“Humacyte”) and carrier (“PBS”). C,D: Alizarin red stain of explantedtissues 21 days after implantation of human elastin that was isolatedaccording to the present invention (“Humacyte”) and carrier (“PBS”).Arrows in panel D indicate areas of possible calcification.

The H&E staining together with an alizarin red staining of explantedtissue from juvenile rats for calcification in FIGS. 4-6 confirms theresults from atomic absorption spectroscopy in FIG. 3 and Table 6regarding the degree of tissue calcification, and shows that humanelastin that is isolated according to the present invention fromnon-frozen tissue does not induce calcification in vivo, using anextremely sensitive implantation model system.

TABLE 6 Calcium levels in explanted samples: Calcium from Explant TissueGroup Elastin source Average St Dev N Rat SubCut Tissue N/A 0.250.160421877 4 PBS carrier N/A 0.49 0.383992568 6 Rat elastin Fresh aorta2.80 3.808000837 2 E56-Humacyte Fresh human aorta 0.47 0.195592829 3E-56 Humacyte Fresh human aorta 0.44 0.081904605 3 gamma E59-HumacyteFrozen human aorta 23.73 6.610771334 3 (frozen) E59-Humacyte Frozenhuman aorta 46.36 17.10482952 3 (frozen) gamma Bovine elastin ElastinProducts Co. 22.26 4.662817486 6 Bovine elastin, Elastin Products Co.28.97 11.63379649 6 gamma Bovine injectable Elastin Products Co. 4.765.485544335 4 Bovine injectable, Elastin Products Co. 9.95 9.181395142 6gamma PBS carrier N/A 0.46 0.172634466 5

Example 6 Isolation of Human Collagen from SMCs on Micro-Carrier Beads

Human vascular smooth muscle cells can also be cultured on micro-carrierbeads in a suspension culture as described herein. During the period ofculturing, the smooth muscle cells replicate on the surface of thebeads, and deposit collagenous extracellular matrix. The collagenousmatrix is then harvested and purified according to the presentinvention. Specific steps in this process are as follows:

1. Culture human smooth muscle cells in a standard culture flask underconditions suitable for growth of the cells, in complete culture mediumcontaining at least 10% serum.

2. Sterilize spinner flask by autoclaving.

3. Weigh out 2.0 g Cytodex-1 micro-carrier beads and mix with 500 mL ofPBS, then sterilize by autoclaving.

4. Pipet 10 mL of micro-carrier bead slurry into spinner flask reactor.

5. Trypsinize vascular smooth muscle cells and seed onto beads inspinner flask a total of 5 million cells in 25 mL of cell growth medium.

6. Culture cells on beads in the presence of DMEM medium containing atleast 10% serum, ascorbic acid (50 mg/L), growth factors such asplatelet derived growth factor (10 ng/mL), basic fibroblast growthfactor (10 ng/mL), epidermal growth factor (3 ng/mL), proline 50 mg/L,glycine 50 mg/L, alanine 20 mg/L, copper sulfate 3 ng/mL

7. Spin the flask at low speed, preferably not more than 10 revolutionsper minute, during culture.

8. Supplement vitamin C twice per week, and replace cultures with freshmedium once per week.

9. After 4-12 weeks of culture of smooth muscle cells on beads, decantoff culture medium supernatant and retain beads contains cells andcollagenous matrix.

10. Digest bead and tissue material in pepsin (0.5 to 2.0 mg/ml pepsinconcentration dissolved in a low pH solution) at 4-20° C. Agitationduring digest will aid the process.

11. Once digestion has completed, centrifuge briefly to remove anyundigested material by filtration, to remove micro-carrier beads.

12. Remove supernatant and raise the pH of the solution to about pH 8.5by slowly adding NaOH to inactivate pepsin.

13. Using HCl, bring the pH of the solution back to about pH 3.5.

14. Clarify collagen solution using diatomaceous earth.

15. Precipitate clarified collagen by adding NaCl to the solution.Precipitate at 4° C. for >24hrs.

16. Collect precipitated collagen by chilled centrifugation at highspeeds for ˜30 minutes.

17. Aspirate supernatant carefully and re-suspend precipitated collagensin ice-cold HCl. Allow for collagen molecules to completely solubilized.

18. Dialyze solution to further purify collagen.

19. Concentrate collagen to desired level.

20. Store this purified collagen in solution.

21. Add sterile-filtered sodium diphosphate solution to concentrated,purified, collagen, until final concentration of about 20-50 mM andabout pH 7.4 is reached. Incubate at 22-37° C. for >24 hours.

The purity of the resultant solubilized product produced as describedherein was compared to other purified human collagens from commercialresources by polyacrylamide gel electrophoresis as shown in FIG. 7. Lane1 shows 20 micrograms of PureCol Human Collagen, Inamed Biomaterials.Lane 2 shows 10 micrograms of collagen derived from human dermalfibroblasts. Lane 3 shows 10 micrograms of human collagen derived fromvascular smooth muscle cells and purified as described in the instantexample. The gel was stained with coomassie blue for protein detection.Typical collagen bands alpha, beta and gamma were present in allsamples. The results in FIG. 7, Lane 3, demonstrate the high purity ofcollagen produced by the instant methods when compared with othersamples of highly purified human collagen.

Example 7 Formulation of Injectable Collagen Material

Collagen can be formulated into an injectable product that can bedelivered to patients. Soluble collagen is collected from engineeredvascular tissue using the procedure contained in Example 1. Precipitatedcollagen is centrifuged and the supernatant removed by aspiration. Theprecipitated collagen is then re-suspended in a physiological salinebuffer solution that is pharmaceutically acceptable (such as 0.9% sodiumchloride solution) that can contain 0.3% lidocaine. The mixture isagitated to ensure uniform mixing, and pH adjusted to approximately 7.0.The volume of re-suspension solution is titrated such that the finalconcentration of collagen is approximately 30 mg/mL. The collagensolution is then dispensed into sterile syringes and packaged in sterilefashion, for clinical applications.

Example 8 Formulation of Injectable Collagen and Elastin CompositeMaterial

[000120] Collagen and elastin can be combined into a composite productthat is injectable.

Soluble collagen is collected from engineered vascular tissue using theprocedure contained in Example 1. Precipitated collagen is centrifugedand the supernatant removed by aspiration. The precipitated collagen isthen re-suspended in a physiological saline buffer solution that ispharmaceutically acceptable (such as 0.9% sodium chloride solution) thatcan contain 0.3% lidocaine. The mixture is agitated to ensure uniformmixing, and pH adjusted to approximately 7.0. The volume ofre-suspension solution is titrated such that the final concentration ofcollagen is approximately 20 mg/mL.

To generate the collagen-elastin composite injectable formulation,elastin is isolated from non-frozen aortic tissue according to Example4. Insoluble elastin isolated after ether extraction is pulverized (withor without a prior freezing step to aid in particle formation) and thenis sieved under sterile conditions to select particles that are lessthan 50 microns. Dry particles are then admixed and suspended withincollagen-containing solution, in order to create the collagen-elastincomposite solution. The collagen-elastin composite is then dispensedinto sterile syringes and packaged in sterile fashion, for clinicalapplications.

Another means by which the elastin may be rendered suitable forinjection is by digestion in pepsin or some other protease with elastaseactivity. Digestion of purified elastin with pepsin at room temperatureor at 37° C. for between 1-5 hours generates elastin fragments ofmolecular weight greater than 100,000. Elastin fragments are thenpurified from residual pepsin utilizing size-exclusion dialysismembranes, and then concentrated to a final concentration ofapproximately 10 mg/mL or greater in a physiologically acceptablecarrier such as 0.9% saline. Suspended elastin fragments are thencombined with precipitated collagen or collagen-containing solution toproduce a final product with a concentration of collagen 20 mg/mL, and aconcentration of elastin 10 mg/mL, in saline with 0.3% lidocaine. Thecollagen-elastin composite is then dispensed into sterile syringes andpackaged in sterile fashion, for clinical applications.

We claim:
 1. A composition comprising isolated human collagen, isolatedhuman elastin and a pharmaceutically acceptable carrier wherein thehuman elastin is substantially insoluble in water with a molecularweight greater than 100 kDa.
 2. The composition of claim 1, wherein theisolated human collagen is derived from engineered vascular tissue. 3.The composition of claim 1, wherein the isolated human collagen isderived from micro-bead culture.
 4. The composition of claim 1, whereinthe isolated human elastin is derived from engineered vascular tissue ornative vascular tissue.
 5. The composition of claim 1, wherein theisolated human collagen has a molecular weight of about 100 to about 500kDa.
 6. The composition of claim 1, wherein the isolated human elastinis cross-linked.
 7. The composition of claim 1, wherein the compositioncomprises about 10-100 mg/ml of isolated human collagen.
 8. Thecomposition of claim 1, wherein the composition comprises about 30 mg/mlof isolated human collagen.
 9. The composition of claim 1, wherein thecomposition comprises about 2 to about 60 mg/ml of isolated humanelastin.
 10. The composition of claim 1, wherein the compositioncomprises about 3 to 30 mg/ml of isolated human elastin.
 11. Thecomposition of claim 1, wherein the composition further comprisesisolated human glycosaminoglycans.
 12. The composition of claim 1,wherein the composition further comprises adipose tissue.
 13. Thecomposition of claim 1 wherein the composition further comprises dermalfibroblasts.
 14. The composition of claim 1, wherein the compositionfurther comprises one or more active agents selected from the groupconsisting of one or more anti-inflammatory agents, tissue formationagents, adipose tissue formation agents, anesthetics, antioxidants,heparin, epidermal growth factor, transforming growth factor,transforming growth factor-β, platelet-derived growth factor, fibroblastgrowth factor, connective tissue activating peptides, β-thromboglobulin,insulin-like growth factors, tumor necrosis factors, interleukins,colony stimulating factors, erythropoietin, nerve growth factors,interferons or combinations thereof.
 15. A dermal or subdermal fillercomprising composition of claim
 1. 16. The composition of claim 1,wherein said elastin is isolated from human non-frozen vascular tissueand wherein the composition does not induce calcification in vivo.
 17. Amethod for soft tissue augmentation in a subject comprising,administering a composition comprising isolated human collagen, isolatedhuman elastin and a pharmaceutically acceptable carrier wherein thehuman elastin is substantially insoluble in water with a molecularweight of above 100 kDa.
 18. The method of claim 17, wherein the softtissue augmentation improves condition selected from the groupconsisting of lines, folds, wrinkles, minor facial depressions, cleftlips, correction of minor deformities due to aging or disease,deformities of the vocal cords or glottis, deformities of the lip,crow's feet and the orbital groove around the eye, breast deformities,chin deformities, augmentation; cheek and/or nose deformities, acne,surgical scars, scars due to radiation damage or trauma scars, andrhytids.
 19. The method of claim 17, wherein the soft tissue is locatedin the pelvic floor, in the peri-urethral area, near the neck of theurinary bladder, or at the junction of the urinary bladder and theureter.
 20. The method of claim 17, wherein the soft tissue augmentationincreases tissue volume.
 21. The method of claim 17, wherein thecomposition is injected into the skin.
 22. The method of claim 17,wherein the composition is injected underneath the skin.
 23. The methodof claim 17, wherein the composition comprising insoluble elastinderived from human vascular tissue does not induce an inflammatory orimmune response and does not induce calcification.
 24. A kit foraugmentation of a soft tissue comprising composition of claim 1, asyringe, a sterile wrapper surrounding said syringe and providing asterile environment for said syringe and any other material/ and orreagents necessary.
 25. The kit of claim 24 wherein said reagentsinclude agents selected from the group consisting of heparin, epidermalgrowth factor, transforming growth factor-alpha, transforming growthfactor-beta, platelet-derived growth factor, fibroblast growth factor,connective tissue activating peptides, β-thromboglobulin, insulin-likegrowth factors, tumor necrosis factors, interleukins, colony stimulatingfactors, erythropoietin, nerve growth factors, interferons, osteogenicfactors and bone morphogenic proteins.